突变
定向进化
计算生物学
质粒
突变体
重叠延伸聚合酶链反应
突变
生物
聚合酶链反应
基因
计算机科学
遗传学
作者
Weilan Shao,Kesen Ma,Yilin Le,Hongcheng Wang,Sha Chen
出处
期刊:Methods in molecular biology
日期:2016-10-06
卷期号:: 497-506
被引量:8
标识
DOI:10.1007/978-1-4939-6472-7_34
摘要
Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.
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